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Hepatocellular carcinoma (HCC) represents a worldwide health matter with a major care burden, high prevalence, and poor prognosis. Its pathogenesis mainly varies depending on the underlying etiological factors, although it develops from liver cirrhosis in the majority of cases. This review summarizes the role of the most interesting soluble factors as biomarkers for early diagnosis and as recommended targets for treatment in accordance with the new challenges in precision medicine. In the premalignant environment, inflammatory cells release a wide range of cytokines, chemokines, growth factors, prostaglandins, and proangiogenic factors, making the liver environment more suitable for hepatocyte tumor progression that starts from acquired genetic mutations. A complex interaction of pro-inflammatory (IL-6, TNF-α) and anti-inflammatory cytokines (TGF-α and -β), pro-angiogenic molecules (including the Angiopoietins, HGF, PECAM-1, HIF-1α, VEGF), different transcription factors (NF-kB, STAT-3), and their signaling pathways are involved in the development of HCC. Since cytokines are expressed and released during the different stages of HCC progression, their measurement, by different available methods, can provide in-depth information on the identification and management of HCC.

Simple Summary The progression of liver disease is accompanied by pathological angiogenesis, a prerequisite for the development of HCC. In this paper, we analyzed the clinical significance of serum angiogenic markers VEGF, Ang-1, Ang-2, angiopoietin receptor Tie1/2, HGF, and PECAM-1 in 62 patients with liver disease, out of which 33 were diagnosed with HCC and 29 with liver cirrhosis without signs of neoplasia. Biomarkers levels were investigated as a function of “Model for End-Stage Liver Disease” (MELD) score and Fibrosis Index (FI). HCC patients showed higher HGF levels than ones with cirrhosis, while high Ang-1 levels appeared to have a protective role in HCC as well as prognostic significance; we also found a strong correlation between HGF levels, Ang-2, and VEGF levels, further supporting their role in tumor angiogenesis. Due to the complexity of angiogenesis and the small size of the study group, further investigations are widely desired especially in the era of immunotherapy and HCC-targeted anti-angiogenic drugs. Abstract Background: Hepatocellular carcinoma (HCC) is a global health problem associated with chronic liver disease. Its pathogenesis varies according to the underlying etiological factors, although in most cases it develops from liver cirrhosis. The disease progression is accompanied by pathological angiogenesis, which is a prerequisite that favors the development of HCC. Aims: This study aims at contributing to our understanding of the role of angiogenic factors in the progression of liver disease. For this purpose, we evaluate the clinical significance of serum angiogenic markers (VEGF, Ang-1, Ang-2, the angiopoietin receptor Tie1/2, HGF, and PECAM-1) first in cirrhotic and HCC patients separately, and then comparing cirrhotic patients with and without HCC. Materials and Methods: We enrolled 62 patients, out of whom 33 were diagnosed with HCC and 29 with liver cirrhosis without signs of neoplasia. Patients underwent venous blood sampling before and after receiving treatments for the diagnosed disease. Serum markers were evaluated using ELISA assays for Tie1 and the Bio-Plex Multiplex system for the remaining ones. Biomarker levels were investigated as a function of clinical scores for disease staging (MELD and Fibrosis Index, FI). Results: In cirrhotic patients, Ang-1 and Ang-2 correlate with MELD (ρAng-1 = −0.73, p = 2E−5) and FI (ρAng-1 = −0.52, p = 7E−3, ρAng-2 = 0.53, p = 3E−3). A reduction of Ang-2 levels (p = 0.047) and of the Ang-2/Ang-1 ratio (p = 0.031) is observed in cirrhotic patients diagnosed with viral hepatitis after antiviral treatments. In HCC patients, Ang-1 negatively correlates with FI (ρ = −0.63, p = 1E−4), and PECAM-1 positively correlates with MELD (ρ = 0.44, p = 0.01). A significant Ang-1 reduction was observed in deceased patients during the study compared to ones who survived (p = 0.01). In HCC patients, VEGF levels were increased after tumor treatment (p = 0.037). Notably, HGF levels in cirrhotic patients with HCC are significantly raised (p = 0.017) compared to that in those without HCC. Conclusions: Our results suggest that serum angiogenic markers, with emphasis on Ang-1/2, can contribute to the development of quantitative tools for liver disease staging and therapy monitoring. The comparison between cirrhotic patients with and without HCC suggests that HGF levels are potentially useful for monitoring the insurgence of HCC after a cirrhosis diagnosis. High Ang-1 levels in HCC patients appear to have a protective role as well as prognostic significance.

The antibody-related immune response is mediated by immunoglobulins (Igs), soluble circulating glycoproteins produced by activated B cells that, upon the recognition of specific epitopes on pathogen surfaces, activate, proliferate, and differentiate into antibody-secreting plasma cells. Although the antibodies are effectors of the humoral immune adaptive response, their overproduction in response to a dysregulated proliferation of clonal plasma cell production in tumoral conditions (i.e., multiple myeloma), enriches the serum and urinary matrices, assuming the crucial role of biomarkers. Multiple myeloma (MM) is a plasma cell dyscrasia characterized by the expansion and accumulation of clonally activated plasma cells in bone marrow, determining the release of high amounts of monoclonal component (MC) that can be detected as intact immunoglobulin (Ig), immunoglobulin fragments, or free light chains (FLCs). The importance of detecting biomarkers for the diagnosis, monitoring, and prognosis of diseases is highlighted by the international guidelines that recommend specific assays for the analysis of intact Igs and FLC. Moreover, a developed assay called Hevylite® allows for the quantification of immunoglobulins that are both involved (iHLC) and not involved (uHLC) in the tumor process; this is a fundamental aspect of following up the patient’s workup and evaluating the progression of disease, together with the treatments response. We here summarize the major points of the complex scenario involving monoclonal gammopathies and MM clinical management in view of advantages derived for the use of Hevylite®.

Objectives The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia. Methods We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs. Results We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia. Conclusion The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.

Oxidative and inflammatory damage are underlying mechanisms in various conditions related to male infertility, including varicocele. Free light chains of immunoglobulins (FLCs) are considered markers of low-grade inflammation in various conditions. Nowadays it is known that they may play enzymatic and proteolytic activities; they may activate complement cascade and mast cells, with crucial role in the development of contact sensibility; they may also inhibit the autonomous signaling ability of the B-cell receptor. Their detection in biological fluids may be useful as biomarker of inflammation or altered immune response. Coenzyme Q10 (CoQ10), a lipidic antioxidant and component of the mitochondrial respiratory chain, is involved in spermatozoa energy metabolism and motility. Therefore, we aimed to evaluate FLCs seminal levels in patients with varicocele in comparison to control subjects and to correlate them with CoQ10 and Total Antioxidant Capacity (TAC) in human semen. A total of 65 patients were enrolled, aged 18-50 years. Semen analysis was performed; patients were divided into three groups: controls (CTRL), 12 normozoospermic patients aged 34 (33-41) years; varicocele (VAR), 29 patients aged 33 (26-37) years; idiopathic (IDIO), 24 oligo-, astheno- and oligoasthenozoospermic patients aged 37 (33.5-40.5) years. FLCs were assayed by turbidimetric method; CoQ10 by HPLC; TAC by spectrophotometric method. Λ FLCs showed a trend towards higher levels in VAR vs CTRL and IDIO. VAR showed a trend towards lower κ FLCs levels vs the other two groups. Comparing κ/λ ratio VAR showed significantly lower levels vs CTRL and IDIO (Mean±SEM 3.18±0.75; 8.28±1.71; 8.90±2.94, respectively; p Our data confirm lower levels of κ/λ ratio in VAR as previously reported and suggest a possible application as clinical biomarkers for male infertility. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Free light chain (FLC) kappa (k) and lambda (λ) consist of low molecular weight proteins produced in excess during immunoglobulin synthesis and secreted into the circulation. In patients with normal renal function, over 99% of FLCs are filtered and reabsorbed. Thus, the presence of FLCs in the serum is directly related to plasma cell activity and the balance between production and renal clearance. FLCs are bioactive molecules that may exist as monoclonal (m) and polyclonal (p) FLCs. These have been detected in several body fluids and may be key indicators of ongoing damage and/or illness. International guidelines now recommend mFLC for screening, diagnosis and monitoring multiple myeloma and other plasma cell dyscrasias. In current clinical practice, FLCs in urine indicate cast nephropathy and other renal injury, whereas their presence in cerebrospinal fluid is important for identifying central nervous system inflammatory diseases such as multiple sclerosis. Increased pFLCs have also been detected in various conditions characterized by B cell activation, i.e., chronic inflammation, autoimmune disease and HCV infection. Monitoring the coronavirus (COVID-19) pandemic by analysis of salivary FLCs presents a significant opportunity in clinical immunology worthy of scientific pursuit.

Oxidative and inflammatory damage underlie several conditions related to male infertility, including varicocele. Free light chains of immunoglobulins (FLCs) are considered markers of low-grade inflammation in numerous diseases. Coenzyme Q10 (CoQ10), a lipidic antioxidant and anti-inflammatory compound, is involved in spermatozoa energy metabolism and motility. We aimed to evaluate FLCs’ seminal levels in patients with varicocele in comparison to control subjects and to correlate them with CoQ10 and Total Antioxidant Capacity (TAC) in human semen. Sixty-five patients were enrolled. Semen analysis was performed; patients were divided into three groups: controls, 12 normozoospermic patients, aged 34 (33–41) years; varicocele (VAR), 29 patients, aged 33 (26–37) years; and idiopathic, 24 oligo-, astheno- and oligoasthenozoospermic patients aged 37 (33.5–40.5) years. FLCs (κ and λ) were assayed by turbidimetric method; CoQ10 by HPLC; TAC by spectrophotometric method. λ FLCs showed a trend toward higher levels in VAR vs. controls and the idiopathic group. VAR showed a trend toward lower κ FLCs levels vs. the other two groups. When comparing κ/λ ratio, VAR showed significantly lower levels vs. controls and idiopathic. Moreover, CoQ10 seminal levels showed higher levels in VAR and idiopathic compared to controls. Data reported here confirm lower levels of κ/λ ratio in VAR and suggest a possible application in personalized medicine as clinical biomarkers for male infertility.

Introduction: The involvement of complement system in the phenotypic expression of systemic sclerosis (SSc) is a debated topic. We aimed to assay complement fractions in SSc patients and to correlate their levels with the clinical course of disease. Key points: 1. CH50 is increased in SSc patients compared to HC; 2. Serum C2 levels are increased in SSc patients compared to HC; 3. CH50 may represent a biomarker of skin and lung fibrosis severity in SSc patients. Method: Complement hemolysis 50% (CH50), C2, C3 and C4 levels have been assessed in 85 SSc patients and 47 healthy controls (HC). Results: SSc patients displayed a statistically significant higher value of CH50 [76.3 U/mL (IQR 65.8–89.4 U/mL) vs. 29.6 U/mL (IQR 24.7–34 U/mL); p < 0.0001] and of C2 [26.1 mg/L (IQR 24.1–32.1 mg/L) vs. 22.7 mg/L (IQR 20.6–24.4 mg/L); p < 0.0001] if compared to HC. Patients with diffuse cutaneous SSc (dcSSc) had higher levels of CH50 than patients with limited cutaneous SSc (lcSSc) [83.6 U/mL (IQR 72.3–102.7 U/mL) vs. 71.3 U/mL (IQR 63.7–83.6 U/mL); p = 0.003]. SSc patients with interstitial lung disease (ILD) had higher CH50 levels if compared to SSc patients without ILD [79.6 U/mL (IQR 68.3–97.4 U/mL) vs. 69.7 U/mL (54.6–85.7 U/mL); p = 0.042]. A positive linear correlation existed between CH50 and the modified Rodnan Skin Score (mRSS) (r = 0.285, p = 0.008) and disease severity scale (DSS) (r = 0.285, p = 0.005); a negative linear correlation was demonstrated between CH50 and the diffusing capacity of carbon monoxide (DLco) (r = −0.252, p = 0.012). In multiple linear regression analysis, only DSS was significant (p = 0.01, beta coefficient 2.446). Conclusions: Our results show an increment of CH50 and serum C2 levels in SSc patients in comparison to HC; we retain that CH50 may represent a biomarker of disease severity and of skin and lung fibrosis in these patients.

Immunoglobulins free light chains (FLCs) are assayable in several biological fluids. Currently, there are no reports on FLCs in seminal plasma. The aims of our study were to investigate the presence and detectability of FLCs in seminal plasma and to evaluate the usefulness of this assay in the diagnostic approach to infertile patients. We enrolled 61 patients aged 18-50 years. Semen analysis was performed. They were divided into four groups: controls-normozoospermic, 10 patients, mean ± SEM age 35 ± 1.5 years; varicoceles (VAR), 18 patients aged 24.3 ± 0.96 years; inflammatory (INF) seminal fluids, 24 patients, aged 38.8 ± 2.2 years; and varicoceles and inflammatory (VAR/INF) seminal fluids, 9 patients, aged 29.5 ± 0.71 years. A trend towards higher λ FLCs levels was evidenced in the INF and VAR/INF groups. κ FLCs were higher in normozoospermic patients with lower levels in VAR, both isolated and associated with inflammatory parameters. This differential pattern of the two types of FLCs reached statistical significance when comparing κ/λ ratio, with significant lower levels in VAR vs controls. This is the first report of FLCs assay in seminal plasma. We can hypothesize that λ FLCs are increased in inflammatory processes, whether κ FLCs seem to be influenced by other molecular mechanisms related to varicocele.

Background: Hepatocellular carcinoma (HCC) is a global health problem associated with chronic liver disease. The pathogenesis of chronic liver disease varies according to the underlying etiological factor, although in most cases it develops from a liver cirrhosis. The worsening progression of liver disease is accompanied by pathological angiogenesis, which is a prerequisite that favors the development of HCC. The aim of this study is to evaluate the clinical utility of circulating angiogenic markers VEGF, Ang-1, Ang-2, the Angiopoietin receptor (Tie1/2), HGF and PECAM-1 to screen early onset patients and to follow the evolution of HCC. Materials and Methods: We enrolled 62 patients; 33 out of 62 subjects were diagnosed for HCC and 29/62 for liver cirrhosis of different etiology without signs of neoplasia. Patients underwent venous blood sampling before and after treatments for VEGF, Ang-1, Ang-2, Tie1, Tie2, HGF and PECAM-1 measurement. Results: Ang-1 and Ang-2 are detectable not only in patients already suffering from HCC but also in cirrhotic patients without signs of cancer. Patients with HCC show higher HGF concentrations than patients with cirrhosis. A significant reduction in serum levels of Ang-2, Ang-2/Ang-1 and Ca 19-9 after DAAs therapy was observed. Moreover, VEGF levels were increased after treatment of HCC. Conclusion: The preliminary study here presented confirms that the mechanism of tumor angiogenesis is very complex and involves a very large number of factors. The integration of different methodologies and multi-marker algorithms is likely to emerge for the early diagnosis of HCC and the monitoring of the risk of relapse.

Myasthenia gravis with antibodies (Abs) against the muscle-specific tyrosine kinase (MuSK) is a rare autoimmune disorder (AD) of the neuromuscular junction (NMJ) and represents a prototype of AD with proven IgG4-mediated pathogenicity. Thanks to the mechanism of Fab-arm exchange (FAE) occurring in vivo, resulting MuSK IgG4 k/λ Abs increase their interference on NMJ and pathogenicity. The characterization of hybrid MuSK IgG4 as a biomarker for MG management is poorly investigated. Here, we evaluated total IgG4, hybrid IgG4 k/λ, and the hybrid/total ratio in 14 MuSK-MG sera in comparison with 24 from MG with Abs against acetylcholine receptor (AChR) that represents the not IgG4-mediated MG form. In both subtypes of MG, we found that the hybrid/total ratio reflects distribution reported in normal individuals, instead, when we correlated the hybrid/total ratio with specific immune-reactivity we found a positive correlation only with anti-MuSK titer, with a progressive increase of hybrid/total mean values with increasing disease severity, indirectly confirming that most part of hybrid IgG4 molecules are engaged in the anti-MuSK pathogenetic immune-reactivity. Further analysis is necessary to strengthen the significance of this less unknown biomarker, but we retain it is full of a diagnostic-prognostic powerful potential for the management of MuSK-MG.

Summary Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways.

Along years, the advent of biological therapy widely modified treatment of rheumatic diseases and other disorders. However, many agents may elicit in anti-drug antibodies (ADAbs) upon consecutive infusions, with a loss of response. For the right strategy of a personalized medicine, the therapeutic monitoring of TNF-α inhibitors and ADAbs represents an important effort in diagnostic-therapeutic pathway, to improve overall patient management and favoring an appropriate clinical approach. A raising number of diagnostic tests have been designed to elucidate the efficacy and/or safety of a specific drug or class of drugs for a targeted patient's group. Our paper reviewed the current understanding of the immunogenicity of biological drugs employed in the treatment of inflammatory diseases underlying the laboratory role.

Polycystic ovary syndrome (PCOS), as systemic disease, is accompanied by different indexes of inflammation. Free light chains of immunoglobulins (FLCs), produced by plasmacells, are released in slight excess for the immune requests, with still poorly defined physiological role but surely they represent a marker of inflammation. In order to evaluate their levels and correlate them with hyperandrogenism, we have studied a group of PCOS patients, age range 18-37 yrs, mean ± SEM body mass index (BMI) 24.1 ± 0.9 kg/m

Background: The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) represents a major threat to human health, which impairs the functionality of several organs. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is indeed highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients’ access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for a fast and non-invasive specimens’ collection, which can be repeated multiple times. Methods: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free-light chains (FLC k and λ) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects. Results: We found that each biomarkers differs significantly between the two groups, with p-values ranging from 10-8 to 10-4. The performance ranking of these markers, shows that λFLC level (p=1.4e-8) is the best-suited candidate to discriminate the two groups, with an accuracy of 0.94 (0.87-1.00 95% CI), a precision of 0.91 (0.81-1.00 95% CI), a sensitivity of 1.00 (0.96-1.00 95% CI) and a specificity of 0.86 (0.70-1.00 95% CI). Conclusion: These results suggest λFLC as an ideal indicator of patient conditions. This is more strengthened in consideration that λFLC half-life (approximately 6 hours) is significantly shorter than the IgA one (21 days): thus λFLC appears displaying the potential to effectively monitor patients fluctuation in real-time.

Free light chains (FLC), considered a biomarker of B cell activity, are frequently elevated in different systemic inflammatory-autoimmune diseases. As systemic sclerosis (SSc) clinical course may be variable, this study aimed to assess FLCs levels in serum and urine of affected individuals, as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of k and λ FLC in serum, significantly higher than HC (p=0.0001) alongside with the mean levels of free k/λ ratio and of the k+λ sum (p=0.0001). In addition, SSc patients had significant higher levels in the urine of free k and of k/λ ratio than HC (p=0.0001). SSc patients with increased k+λ sum in serum showed a statistically higher levels of erythro-sedimentation rate (p=0.034), C-reactive protein (p=0.003), higher DAI (p=0.024) and DSS (p=0.015) than SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free k and DAI (r=0.29, p=0.014). SSc patients with increased free k in urine had statistically higher DAI (p=0.048) than SSc patients with normal level of free k. The significant increased levels of FLC correlated with disease activity in SSc patients. Our results strengthen the role of FLC as useful biomarkers in clinical practice to early diagnosis and monitor disease activity with an improvement of SSc patients’ management.

Background and Aims Direct‐acting antivirals (DAAs) usually lead to improvement/remission of cryoglobulinemic vasculitis (CV), although symptoms may persist/recur after a sustained virological response (SVR). We evaluated hematological and genetic markers in patients with HCV‐SVR vasculitis with and without persisting/recurring symptoms to early predict the CV outcome. Approach and Results Ninety‐eight patients with HCV‐CV were prospectively enrolled after a DAA‐induced SVR: Group A: 52 with complete clinical response; Group B: 46 with symptom maintenance/recurrence. Monoclonal B‐cell lymphocytosis, t(14;18) translocation, and abnormal free light chains κ/λ ratios were detected by flow cytometry or nested‐PCR or nephelometry in 4% Group A versus 17% Group B (P = 0.04) patients, 17% Group A versus 40% Group B patients (P = 0.02), and 17% Group A versus 47% Group B (P = 0.003) patients, respectively. At least 1 out of 3 clonality markers was altered/positive in 29% of Group A versus 70% of Group B patients (P

Cryoglobulins consist of serum immunoglobulins that precipitate below 37°C and resolubilize upon warming. The clinical triad of cryoglobulinemia usually includes purpura, weakness, and arthralgia. Cryoglobulinemic syndrome, clinically defined as a systemic vasculitis, is associated with chronic infection with hepatitis C virus (HCV) and autoimmune disorders and can evolve into B-cell malignancies. While the current literature about HCV-associated cryoglobulinemia is not very limited, little is known about the immunologic and serologic profiles of affected patients. Therefore, comprehension of the pathogenetic mechanisms underlying cryoprecipitation could be very helpful. Due to the persistence of viral antigenic stimulation, biomarkers to use after the worsening progression of HCV infection to lymphoproliferative and/or autoimmune diseases are widely needed. Laboratory methods used to detect and characterize low concentrations of cryoprecipitates and immunotyping patterns could improve patient management. The most critical factor affecting cryoglobulin testing is that the pre-analytical phase is not fully completed at 37°C.

Summary Circulating free light chains (FLCs), considered biomarkers of B cell activity, are frequently elevated in patients affected by systemic inflammatory autoimmune diseases. As the systemic sclerosis (SSc) clinical course can be variable, this study is aimed at evaluating FLCs levels in affected individuals as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of κ and λ FLC in serum significantly higher than HC (p = 0.0001) alongside the mean values of free κ/λ ratio and κ + λ sum (p = 0.0001). SSc patients showed increased free κ in urine with a κ/λ higher than HC (p = 0.0001). SSc patients with increased κ + λ in serum showed that erythro-sedimentation rate (p = 0.034), C-reactive protein (p = 0.003), DAI (p = 0.024) and DSS (p = 0.015) were higher if compared to SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free κ and DAI (r = 0.29, p = 0.014). In addition, SSc patients with increased free κ in urine had higher DAI (p = 0.048) than SSc patients with normal κ levels. Our results strengthen the role of serum FLC as useful biomarker in clinical practice to early diagnosis and monitor disease activity, showing for the first time that also urine FLC levels correlated with disease activity in SSc patients.

Metabolic diseases are associated with a higher risk of a severer coronavirus disease 2019 (COVID‐19) course, since fatty liver is commonly associated with metabolic disorders, fatty liver itself is considered as a major contributor to low‐grade inflammation in obesity and diabetes. Recently a comprehensive term, metabolic (dysfunction) associated fatty liver disease (MAFLD), has been proposed. The hepatic inflammatory status observed in MAFLD patients is amplified in presence of severe acute respiratory syndrome coronavirus 2 infection. Intestinal dysbiosis is a powerful activator of inflammatory mediator production of liver macrophages. The intestinal microbiome plays a key role in MAFLD progression, which results in non‐alcoholic steatohepatitis and liver fibrosis. Therefore, patients with metabolic disorders and COVID‐19 can have a worse outcome of COVID‐19. This literature review attempts to disentangle the mechanistic link of MAFLD from COVID‐19 complexity and to improve knowledge on its pathophysiology.

Background Although metabolic associate fatty liver disease (MAFLD) is associated with obesity, it can also occur in lean patients. MAFLD is more aggressive in lean patients compared to obese patients, with a higher risk of mortality. Specific biomarkers to diagnose differentially lean or overweight MAFLD are missing. Histones and nucleosomes are released in the bloodstream upon cell death. Here, we propose a new, fast, imaging and epigenetics based approach to investigate the severity of steatosis in lean MAFLD patients. Results A total of 53 non-obese patients with histologically confirmed diagnosis of MAFLD were recruited. Twenty patients displayed steatosis grade 1 (0–33%), 24 patients with steatosis grade 2 (34–66%) and 9 patients with steatosis grade 3 (67–100%). The levels of circulating nucleosomes were assayed using enzyme-linked immunosorbent assay, while individual histones or histone dimers were assayed in serum samples by means of a new advanced flow cytometry ImageStream(X)-adapted method. Circulating nucleosome levels associated poorly with MAFLD in the absence of obesity. We implemented successfully a multi-channel flow methodology on ImageStream(X), to image single histone staining (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2). We report here a significant depletion of the levels of histone variants macroH2A1.1 and macroH2A1.2 in the serum of lean MAFLD patients, either individually or in complex with H2B. Conclusions In summary, we identified a new circulating histone signature able to discriminate the severity of steatosis in individuals with lean MAFLD, using a rapid and non-invasive ImageStream(X)-based imaging technology.

In the natural history of SARS-CoV-2 infection, liver injury is frequent but quite mild and it is defined as any liver damage occurring during disease progression and treatment of infection in patients with or without pre-existing liver diseases. The underlying mechanisms for hepatic injury in patients with COVID-19 are still unclear but the liver damage in SARS-CoV-2 infection seems to be directly caused by virus-induced cytopathic effects. In this review, we will summarize all data of updated literature, regarding the relationship between SARS-CoV-2 infection, acute response and liver involvement. An overview will be given on liver injury, liver transplant and the possible consequences of COVID-19 in patients with pre-existing liver diseases.

It is known that GH, directly or via IGF-1 activity, influences many aspects of immune response; both humoral and cellular branches are modulated, but no data are available about complement function. CH50 is a screening assay for the activation of the classical complement pathway and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). On the other hand, a low inflammation is present in GH deficiency (GHD) and it is related to cardiovascular risk of such situation, also in partial forms.Therefore, we performed a cohort study in a group of adult GHD patients evaluating CH50 levels to further explore the pattern of inflammatory markers in this condition.We included 17 patients with total GHD (GH peak after stimulation with GHRH+arginine

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete + partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome.

We analyzed the prognostic impact of levels of free light chains (FLC) in 106 patients with DLBCL, selecting 61 patients with a monoclonal (M) protein in serum, and 45 patients without a M protein as an IPI-matched control group. Patients with a M protein had higher levels of FLC, but these were not of prognostic significance in this group. The presence of a M protein nullified associations of κ-FLC with several laboratory parameters indicating immune system activation observed in patients without a M protein. Patients without M protein and κ-FLC50 mg/L had a significant inferior event-free survival (

Biological functions of immunoglobulin-free light chains (FLCs), other than in chronic inflammatory diseases, are still poorly defined; the field of insulin resistance (IR) has not been investigated, despite the strict relationships with oxidative stress (OS) and inflammation. Therefore, we evaluated FLCs levels and their relationships with metabolic parameters in adult growth hormone deficiency (GHD) and metabolic syndrome (MetS), both characterized by IR. One hundred subjects were enrolled: group A, patients with GHD [n =31, 24-69 years, mean ± SEM body mass index (BMI) 26.8 ± 1.5 kg/m2 ]; group B, patients with MetS (n = 29, 21-70 years, BMI 31.9 ± 1.3); group C, controls (N = 40, 21-62 years, BMI 21.6 ± 1.1). Groups were matched by age range and, for patients, by BMI. Morning blood sample was collected for metabolic parameters and FLCs, assessed by turbidimetric assay. GHD patients show levels of FLCs significantly higher than MetS and controls (mean ± SEM κ 37.21 ± 6.97, 15.27 ± 0.86, 12.34 ± 0.85 mg/l; λ 19.44 ± 2.61, 11.78 ± 0.72 and 11.67 ± 0.77 mg/l; κ/λ ratio 1.77 ± 0.13, 1.38 ± 0.09; and 1.10 ± 0.06, respectively); only κ were higher in MetS versus controls. Therefore, the ratio showed progressive declining values in GHD versus MetS versus controls. Our data show increased FLCs levels in GHD and MetS, with the highest values in the former. Both conditions show OS, but with different molecular patterns. FLCs may contribute to chronic inflammation, leading to OS, and cardiovascular complications of GHD. Prognostic and therapeutic implications require further investigation. © 2018 BioFactors, 44(5):480-484, 2018.

In previous studies, we have shown that metabolic syndrome (MetS) and adult growth hormone deficiency (GHD) share different biochemical features, such as insulin-resistance (IR) but differ in antioxidant pattern. On the other hand, immunoglobulin free light chains (FLCs) are molecules with biological functions which are still incompletely known. In order to compare different populations with IR and, consequently, classify different phenotype of IR, we have evaluated FLCs and Total Antioxidant Capacity (TAC) in 3 groups of patients with IR due to different diseases: group A, patients with GHD (n=31, 24-69 ys, mean±SEM BMI 26.8±1.5 kg/m2); group B, patients with MetS (n=29, 29-70 ys, mean±SEM BMI 31.9±1.3 kg/m2); group C hyperferritinaemia (HF) (n=33, 28-78 ys, mean±SEM BMI 31.1±0.99 kg/m2). All patients underwent an oral glucose tolerance test; HOMA-IR was calculated; moreover, a morning blood sample was collected for evaluation of FLCs (assessed by turbidimetric assay) and TAC (assessed by spectrophotometric method, using the system H2O2-metmyoglobin, and expressed as Lag, latency time before the appearance of the radical form of the chromogen ABTS). HOMA-IR, as expected, was high in all three groups (group A 3.13±0.43, group B 3.16±0.28, group C 4.62 ±0.51), significantly higher in group C vs the other two groups. The parameters of inflammation and oxidative stress were significantly different in group A vs the other two groups (FLCs k 36.2±7.0 mg/l vs 15.4±0.9 in group B and 17.8±8.0 in group C; FLCs lambda 19.4±2.6 vs 11.3±0.7 in group B and 12.7±7.3 in group C; Lag 65.0±5.3 sec vs 82.9±6.2 in group B and 83.9±5.4 in group C). These data confirm that different mechanisms of oxidative stress and inflammation underlie these three subtypes of IR, and could be used to classify different phenotypes of these complex syndrome.

The study of free light chains (FLCs) has grown as area of enormous interest for many clinicians with the aim of disclosing the exact biological role and potential use of FLCs in the clinical routine. Moreover, the attention given to immunological functions of FLCs has sparked a new light into their pathogenic contribution in different chronic autoimmune-based inflammatory diseases. The release of intracellular antigens following cell death or ineffective clearance of apoptotic debris, modification of self-antigens, and molecular mimicry may trigger the production of immunoglobulins after activation and polyclonal expansion of B cells, by which FLCs are released. The discovery of polyclonal FLCs as potential biomarkers started with the observation of their increased concentrations in a variety of biological fluids related to patients with autoimmune diseases. This review deals with the use of polyclonal FLCs for identifying severity and monitoring outcome after treatment in some autoimmune diseases, namely systemic lupus erythematosus, myasthenia gravis, systemic sclerosis, rheumatoid arthritis and Sjögren's syndrome, as supported by the fact that levels of FLCs correlate with both B cell activation markers and other specific markers of disease activity. In a near future, following the evidence shown, FLCs might probably work as early prognostic markers of severity and also as indicators of response to treatment or early assessment of relapse in selected autoimmune diseases.

The ongoing outbreak of coronavirus disease 2019 (COVID-19), which impairs the functionality of several organs, represents a major threat to human health. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients’ access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for the fast and non-invasive collection of specimens and can be repeated multiple times. Methods: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free light chains (kFLC and λFLC) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects. Results: We found that each biomarker differs significantly between the two groups, with p-values ranging from 10−8 to 10−4. A Receiving Operator Curve analysis shows that λFLC level is the best-suited candidate to discriminate the two groups (AUC = 0.96), with an accuracy of 0.94 (0.87–1.00 95% CI), a precision of 0.91 (0.81–1.00 95% CI), a sensitivity of 1.00 (0.96–1.00 95% CI), and a specificity of 0.86 (0.70–1.00 95% CI). Conclusion: These results suggest λFLC as an ideal indicator of patient conditions. This hypothesis is strengthened by the consideration that the λFLC half-life (approximately 6 h) is significantly shorter than the IgA one (21 days), thus confirming the potential of λFLC for effectively monitoring patients’ fluctuation in real-time.

Seminal plasma is a complex fluid with various components (proteins, enzymes, macro-and microelements, lipids and nutrients) and its role is fundamental for spermatozoa motility, viability and fertilizing capacity maintenance. Many molecules have been measured in seminal plasma to explore some secretion functions of male accessory glands, but effects of biochemical components in human seminal plasma are still debated. Immunoglobulin-free light chains (FLCs) κ and λ are produced by plasma cells in slight excess for the need of immune response and are therefore assayable in blood and in other biological fluids, such as urine, saliva, liquor and synovial fluid. Recently, different biological functions have been attributed to these molecules, suggesting that they are not just a secondary product of immunoglobulin synthesis. No data are reported about presence of FLCs in seminal plasma and their role in physiology of male reproductive system and/or in pathophysiology of infertility. The aims of our study were to investigate the presence and detectability of FLCs in seminal plasma and to evaluate the usefulness of this assay in the diagnostic approach to infertility patients. We enrolled 32 patients aged 19-40 ys, affected by primary infertility; among them, 7 were normospermic (mean±SEM concentration 100.0±16.0 *106/ml; progressive motile forms 39.1±4.9%; normal forms 45.3±4.5%), 25 were oligo- and/or asthenoteratospermic (mean±SEM concentration 23.8±5.4*106/ml; progressive motile forms 19.3±4.1%; normal forms 36.05±2.7%); moreover, 17 patients presented II-IV degree varicocele (VAR) according to Dubin-Amelar classification by Doppler technique, the remaining 15 patients did not present varicocele (NO-VAR). After abstinence for 3-5 days, semen samples were collected. FLCs concentrations were assayed by turbidimetric method. Standard semen analysis was performed according to WHO laboratory manual for the examination and processing of human semen, fifth edition, 2010. As main results, independently from sperm count, a significantly difference was observed concerning FLCs concentrations, with higher levels of k and k/λ ratio in NO-VAR vs VAR patients (mean±SEM k 36.4±13.2 vs 17.7±9.0 g/l and 7.7±2.9 vs 2.65±0.7, respectively; p

Background HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC). Methods Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures. Results Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test. Conclusions Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naive HCV-patients without symptoms of extrahepatic manifestations.

CH50 is a screening assay for the activation of the classical complement pathway, the immunoglobulins-mediated one, activated in several inflammatory diseases. Adult growth hormone deficiency (aGHD) is recognized as a chronic inflammatory condition, although poorly evaluated under the profile of inflammatory biomarkers. The aim of this case-control observational study is to analyze CH50 and immunoglobulins G (IgG) subclasses production in aGHD, comparing this condition to healthy controls.38 subjects were included and divided as follows: aGHD (n = 18, 6 females and 12 males); healthy controls (n = 20, 10 females and 10 males). GHD was diagnosed with dynamic test using Growth Hormone-Releasing Hormone (GHRH 50 μg i.v. + arginine 0,5 g/Kg), with a peak GH response < 9 μg/L when BMI was 30 kg/m2. The two groups were evaluated for hormonal and metabolic parameters, CH50 and IgG subtypes.IgG1 and IgG2 were significantly higher in controls than in aGHD, while IgG3 and IgG4 showed a trend to higher levels in controls, although not significant. Furthermore, CH50 levels were significantly higher in aGHD.These data substantiate the hypothesis of a dyscrasia in IgG subclasses production in aGHD. As IgG levels decrease, CH50 levels do not.

Clinical spectrum of hepatitis C virus (HCV)-related cryoglobulinemia varies from an asymptomatic presentation to severe vasculitis and lymphoma. A recent study in HCV-negative patients suggests that low cryoglobulins (CGs) levels are responsible for severe renal and neurological complications. The aim of this study was to identify a panel of serological biomarkers associated with low levels of CGs in HCV-positive patients. We studied a population of 79 untreated patients with chronic HCV infection: 13 naive patients without CGs; 28 patients with asymptomatic mixed cryoglobulinemia (MC) and low levels of CGs (16/28 with polyclonal type III and 12/28 with microheterogeneous type III CGs); 38 patients with symptomatic MC and high levels of type II CGs. Serum samples were collected and examined for rheumatoid factor (RF) IgG and IgM, free light chains (FLCs) and C3 and C4 complement components. We found that RF-IgG and IgM, free k chains and k+λ were increased while C4 component was reduced, both in symptomatic and asymptomatic patients. Our results suggest that, even in absence of MC symptoms, the low levels of CGs may represent a trigger of activation for immune system in course of HCV infection. The identification of a correlated biomarkers panel could improve the clinical management of these patients and pave the way for target treatment strategies. © 2018 BioFactors, 45(3):318-325, 2019.

Background Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree. Methods Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns. Results After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate. Conclusion Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia.

Background. Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity. Subjects and Methods. We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution. Results. We found a statistically significant increase in free κ chains in both AChR- and MuSK-MG patients, while free λ chain levels were increased only in AChR-MG. We also observed a significant reduction of both free κ and λ chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment. Conclusions. From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.

AimHumoral immunity and B cells are thought to play an important role in the pathophysiology of the systemic sclerosis (SSc). The production of free light chains (FLC) of immunoglobulins is abnormally high in several pathological autoimmune conditions and reflects B cell activation. Furthermore, FLCs demonstrated different biological activities including their capability to modulate the immune system, proteolytic activity and complement cascade activation. The aims of this study are to determine the FLC levels in patients with SSc compared with healthy controls (HC) and to study their possible association with organ involvement and disease characteristics.MethodsSixty-five patients with SSc and 20 HC were studied. Clinical and immunological inflammatory characteristics were assessed for all the patients with SSc. κ-FLC and λ-FLC, interleukin 6 (IL-6) and B cell activating factor levels were measured.ResultsThe mean serum κ-FLC levels and FLC ratio were significantly higher in patients with SSc compared with HC, while the serum λ-FLC levels were comparable.The levels of FLC were comparable in patients with diffuse skin disease and limited skin involvement, while κ-FLC levels were increased in patients with restrictive lung (forced vital capacity (FVC) ConclusionsFLC levels are elevated in SSc and high levels are associated with lung involvement and with a higher degree of inflammation, supporting a possible role of B cell activation in the pathophysiology of the disease.

HCV is a hepatotropic and lymphotropic virus and is the most frequent cause of "benign" mono-oligoclonal B-lymphocyte proliferation, observed in mixed cryoglobulinemia (MC). The study aims to investigate the presence, prevalence and characteristics of the subclasses of cryoglobulins in HCV-patients to look for a relationship with MC. Fifty HCV-infected patients with cryoglobulins were enrolled. IgG subclasses were characterized in cryoprecipitate, and serum IgG and IgM Rheumatoid Factor (RF) were determined. Patients were stratified into two subgroups according to the presence of IgG3 subclass. Differences were observed in supernatant IgM, IgG3-positive and IgG3-negative patients with a higher IgM concentration in the IgG3-negative cohort (p=0.03). Higher IgM-RF was detected in the IgG3-negative group (p=0.01). IgG3-positive group showed higher IgG-RF compared to the IgG3-negative group (p